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Objective To investigate the protective effect of hepatocyte growth factor (HGF) on cultured Sprague-Dawley rat cortical neurons injured through hypoxia/reoxygenation.Methods Primary cultured cerebral cortical neurons were isolated from newborn rots.Neurons were pre-incubated with different concentrations (15,30 and 60 μg/L) of HGF.The cell viability was detected by MTT.Apoptosis was measured by Hoechst 33258 staining and flow cytometer.Lactate dehydrogenase (LDH) and caspase-3 activity were determined by colorimetry.Results Compared with normal group,hypoxia/reoxygenation treatment significantly decreased cell viability,increased LDH activity and the percentage of apoptotic cells.Pretreatment of HGF for 12 h could remarkably reverse the decrease of cell viability and the increase of apoptosis rate in neurons induced by hypoxia/reoxygenation treatment.HGF pre-treatment also attenuated the activity of LDH and caspase-3 in a dose-dependent manner.The effects of HGF could be inhibited by a special PI3K/Akt pathway inhibitor,LY294002.Condusion HGF could attenuate rat cortical neuron injury induced by hypoxia/reoxygenation.The neuroprotective effect of HGF may be related to activating PI3K/Akt pathway,and further suppressing the expression of caspase-3.
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BACKGROUND:FGF2,TGFβ/activin/nodal and IGF signal pathways are necessary for keeping functions of human embryonic stem calls,but there was no report concerning whether addition of exogenous basic fibroblast growth factor,transforming growth factor β and insulin can maintain self-renewal of human embryonic stern cells.OBJECTIVE:To establish a feeder layer-and serum-free culture system of human embryonic stem cells.DESIGN,TIME AND SETTING:The cytological in vitro study was performed at the Kunming Animal Institute of Chinese Academy of Sciences from September 2007 to February 2009.MATERIALS:Pregnancy 12.5 or 13.5-day ICR strain mica (clean grade) were provided by the Animal Center of Kunming Medical College.Human embryonic stem cell line BG02 was purchased from Bresagen,USA.METHODS:BG02 cells were digested,centrifuged,resuspended in feeder layer-free medium,and then incubated for 5-7 days.Differentiated human embryonic stem cells were removed.Dispase was added for digestion.Samples were cut into blocks,centrifuged,resuspended,and then incubated at 1:3 in a 4-well plate coated with laminin in feeder layer-free and serum-free medium,supplemented with 80% DMEM/F12,20% KSR,2 mmol/L glutamine,1% non-essential amino acid,0.1 mmol/L β-mercaptoethanol,insulin-transferrin-selenium (ITS) (×1),10~6 U/L penicillin,100 mg/L streptomycin,4 μg/L basic fibroblast growth factor (bFGF) and 0.12 μg/L transforming growth factor-β1 (TGF-β1).ICR fetus mica were sterilely obtained to culture mouse embryonic flbroblasts by tissue pancreatin digestion method.These cells were incubated in a 6-well plate coated with 0.1% gelatin.Human embryonic stem cell line BG02 cultured on mouse embryonic fibroblasts feeder layers was transferred to laminin-coated plates in serum-free medium containing bFGF,TGFβ1 and ITS.MAIN OUTCOME MEASURES:The morphology of BG02 cells in feeder layer-and serum-free condition was observed.The specific molecular markers of human embryonic stem cells were determined by immunohistochemical staining.The karyotype and differentiation ability in vitro of BG02 cells were analyzed.The differences in the proliferation and the differentiated rate of BG02 clumps in feeder layer-and serum-free condition or mouse embryonic fibroblast feeder layer condition were compared.RESULTS:BG02 calls had been continuously cultured for at least 20 passages in feeder layer-and serum-free culture condition.BG02 clones in this culture system had typical human embryonic stem cell morphology.BG02 cells all expressed SSEA-4,SSEA-3,TRA-1-60,TRA-1-81,Oct-4,but did not express SSEA-1.After 20 days of culture,BG02 cells had the capacity to differentiate into derivatives of embryonic endoderm,mesoderm,and ectoderm.Differentiated cells could express alpha-fetoprotein,nestin,α-actin.At passage 20,call karyotype was normal (46XY).The growth of BG02 cells cultured in feeder layer-and serum-free condition was more slowly compared with mouse embryonic fibroblast feeder,and doubling time was significantly prolonged (P ＜ 0.05).Differentiation rate of the colonies was significantly increased (P ＜ 0.05).CONCLUSION:A feeder layer-and serum-free condition for culture of BG02 cells has been established.The addition of bFGF,TGFβ1 and ITS can maintain the self-renewal of BG02 cells.
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BACKGROUND: Whether human embryonic stem cells (hESCs) cultured on different feeder layers can maintain identical or similar characteristics remains unclear.OBJECTIVE: To compare the characteristics of hESCs cultured on human- and mouse- origin feeder layers.DESIGN, TIME AND SETTING: The in vitro cytology observation was performed at the Kunming Institute of Zoology, Chinese Academy of Sciences between September 2007 and February 2009.MATERIALS: Two ICR pregnant mice with 12.5-13.5 embryonic days were provided by Animal Center of Kunming Medical College. Immortalized human adult fibroblast (HAFi) cell line was presented by School of Medicine, Johns Hopkins University (USA). hESCs line was provided by Kunming Institute of Zoology, Chinese Academy of Sciences.METHODS: Mouse embryonic fibroblasts (MEF) were harvested from ICR mice by trypsinization method. HAFi were conventionally cultured. After γ ray treatment, two kinds of cells were incubated on 6-well gelatin-coated plate with density of 2.5×10~4/cm~3. hESCs were cultured on HAFi or MEF feeder cells containing β-mercaptoethanol DMEM/F12 and basic fibroblast growth factor.MAIN OUTCOME MEASURES: Morphology, expressions of specific molecular markers, Oct-4 positive rate, as well as cell doubling time of hESCs cultured by two methods were compared.RESULTS: ①BG02 cells on MEF and HAFi shared the similar morphology and characteristic pluripotency markers, which expressed SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and Oct-4, but not SSEA-1. However, the proportion of positive Oct-4 cells in hESCs colonies maintained on MEF was lower than that of HAFi (P < 0.05) with shorter doubling time (P < 0.05).CONCLUSION: hESCs cultured on MEF and HAFi represent some differences in the growth and pluripotency characteristics.
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Teaching is an art,which requires teachers to have high profession level and better ability of language expression.To stress teaching techniques is the request of higher medical science education.It is worth summarizing and thinking seriously.This article summarized several aspects of teaching techniques,essence.In addition,it explained the significance of teaching techniques.
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AIM: To investigate the effect of non-activated or activated polymorphonuclear leukocytes(PMN) on washed platelet aggregation. METHODS: Born's method was used to determine platelet aggregation.RESULTS: non-activated PMN (5?10 9 cells/L) significantly suppressed washed platelet aggregation induced by ADP or arachidonic acid. Aspirin enhanced this inhibition. N-formyl-methiongl-leucy-phenylalanine (fMLP)-or platelet-activating factor (PAF)-stimulated PMN strongly induced platelet aggregation, and the induction effect of PMN suspension was more active than that of PMN supernatant. Aspirin had no significant inhibitory effect on platelet aggregation induced by fMLP-or PAF-activated PMN. CONCLUSIONS: Different conditions of PMN (activated or non-activated) had the nearly opposite action on normal platelet reactivity. Briefly, non-activated-PMN inhibited platelet reactivity, whereas activated PMN stimulated it.
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Objective To explore the effects of notoginsenoside-Rg_1 on brain-derived neurotrophic factor(BDNF) protein in cerebrum cortex of MCAO/R injury,as well as to investigate whether notoginsenoside-Rg_1 can up-regulate the protein content of BDNF of the positive neurons or the amount of the po-sitive neurons.Methods Adult male SD rats(60) were randomly divided into model group,notoginsenoside-Rg_1 high,middle,and low dose(200,100,50 mg/kg) groups,and the positive control(Nimodipine,1 mg/kg) group.All drugs were given once a day by ip till the mouse was killed.The focal cerebral(ischemia-)reperfusion model was made with thread-occluded method.Their frozen brain tissue were sliced(into) section of 12 ?m thickness.The four rats were randomly taken from each groups to be treated as specimens after surgical handle in 1,3,and 7 d.The slices were according to the immunohistochemical ABC techniques.The protein content of BDNF of the positive neurons and the amount of the positive neurons in cerebrum cortex of rat were observed and counted by HPIAS—1000 analytic system,and the nervous deficit symptoms after the cerebral ischemia were observed.Results Comparing with the model group,all notoginsenoside-Rg_1 treated groups obviously improved some nervous deficit symptoms of cerebral ischemia-reperfusion rats,and increased the protein content of BDNF and the amount of the positive neurons in the cerebrum cortex of model rats(P
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AIM: To investigate the effects of different concentrations of thrombin, arachidonic acid (AA) and aspirin on leukocyte-platelet adhesion. METHODS: The methods of Hamburger et al and Shen Zhi-qiang et al were used to study the adhesion between platelets and leukocytes in rats. RESULTS: 50 U/L of thrombin markedly stimulated the binding between platelets and leukocytes; the efficacy of thrombin increased with its concentration and produced the maximum effect at 300 U/L. But the adhesion rate decreased while the concentration of thrombin was more than 300 U/L. 25 ?mol/L of AA significantly enhanced the binding of platelets to leukocytes; the efficacy of AA increased with its concentration and obtained the maximum effect at 100 ?mol/L. The adhesion rate, however, decreased while the concentration of AA was more than 100 ?mol/L. Aspirin could inhibit thrombin-or AA-induced adhesion between platelets and leukocytes. CONCLUSION: It is suggested that thrombin and AA, in a certain range of concentrations, concentration-dependently induced the binding of platelets to leukocytes; the adhesive rate, however, decreased as the concentration of the above inducers increased. Aspirin could inhibit platelet-leukocyte adhesion stimulated by thrombin and AA.